Effect Of NOXs-TRPM2 On 5-HT Induced Contractile Reactivity Of Pulmonary Artery Branch In Chronic Hypoxic Pulmonary Hypertension Rats

Pulmonary hypertension（PH）is a serious disease with high mortality.Almost all types of pulmonary hypertension have endothelium-dependent vasodilation damage and systolic hyperresponsiveness.Serotonin（5-HT）is a powerful pulmonary vasoconstrictor with elevated plasma levels in patients with PH.In the animal model of PH,it was also confirmed that chronic hypoxia could enhance the pulmonary vasoconstriction induced by 5-HT.Previous studies have shown that the production of 5-HT can activate the corresponding receptors,resulting in the activation of nicotinamide adenine dinucleotide phosphate（NADPH）oxidase,and then produce hydrogen peroxide（H₂O₂）,resulting in the contraction of systemic circulatory arteries and vascular smooth muscle.Transient receptor potential melastatin 2（TRPM2）is a redox sensitive cation channel,adjusted by many factors,including[Ca²⁺]_i,synergistic promotion of adenosine diphosphate ribose（ADPR）and H₂O₂.H₂O₂ and oxidative stress can indirectly activate TRPM2 by promoting nuclear polymerization PARPs formation and activity of NAD enzymes（NADase）in mitochondria.Antioxidants or drugs targeting specific sources of reactive oxygen species（ROS）have been considered potential treatments for PH.In the early laboratory studies,it was found that the pulmonary artery of rats showed a significantly increased dose-dependent contraction of 5-HT during PH,which could be attenuated by NOX inhibitors.In addition,the manifestations of pulmonary arteries（PAs）were significantly different from those of small pulmonary arteries（sPAs）in the early stage of PH.There were no obvious pathological changes in PAs in the early stage,while sPAs showed severe myalization earlier.In terms of function,it also showed significant differences between PAs and sPAs,such as different vascular reactivity to a variety of vasoactive substances,significant differences in proliferation ability and so on.In this study,we established a chronic hypoxic pulmonary hypertension（CHPH）model and pretreated sPAs with different NOXs inhibitors and TRPM2 inhibitors to observe the changes of dose-dependent contraction induced by 5-HT,to study the role of NOXs-TRPM2 in sPAs contractile response induced by 5-HT in CHPH rats,and to explore the main NOX subtypes involved in this process and the signal pathways that may be involved in 5-HT-induced vasoconstriction.Objective:To observe the effect of NOXs-TRPM2 inhibitor on 5-HT-induced sPAs contraction in chronic hypoxia-induced PH model rats,and to explore the role of NOXs-TRPM2 pathway in 5-HT-induced sPAs contraction in CHPH rats.Methods:（1）The establishment of chronic hypoxia-induced PH model was evaluated by hemodynamic measurement of right ventricular systolic pressure（RVSP）and right ventricular mass index（RVMI）.（2）Microvascular tension was used to detect vascular tension to observe the contractile effect of sPAs induced by 5-HT in rats.（3）The changes of vascular tension and Ca²⁺signal in smooth muscle cells of sPAs were recorded synchronously by laser confocal tension detection system.Results:（1）After three weeks of chronic hypoxia induction,RVSP and RVMI increased significantly in rats,suggesting that the model of pulmonary hypertension was successfully established.（2）The contractile reactivity of sPAs induced by KCl was significantly higher than that in PAs,but there was no significant difference between normal rats and CHPH rats.（3）Compared with the control group,there was no significant difference in KCl induced vasoconstriction in sPAs of normal rats,which was pretreated with NOX1/2/4 inhibitors ML171,gp91 ds-tat and GKT.（4）TRPM2inhibitors ACA and 2-APB pretreated normal rats with sPAs inhibited KCl-induced vasoconstriction in a dose-dependent manner,suggesting that appropriate doses should be selected to ensure that their inhibitory effects on sPAs are specific.（5）The contractile response induced by 5-HT had almost no effect on sPAs in normal rats,but was significantly enhanced in CHPH rats.（6）The role of NOXs in 5-HT-induced sPAs contraction:a.ML171,gp91 ds-tat and GKT preconditioning inhibited sPAs contraction induced by 5-HT in normal rats,and there was no significant difference among the three groups.b.Pretreatment with ML171,gp91 ds-tat and GKT inhibited 5-HT-induced sPAs contraction in CHPH rats,and the inhibitory amplitude was ML171/GKT>gp91 ds-tat.c.ML171 and GKT preconditioning inhibited the contraction of sPAs and the increase of Ca²⁺signal in smooth muscle cells（F/F0）induced by 5-HT in CHPH rats,and the inhibitory amplitude of ML171 was higher than that of GKT.（7）The role of TRPM2 in sPAs contraction induced by 5-HT:a.ACA and 2-APB preconditioning inhibited sPAs contraction induced by 5-HT in normal rats,and the inhibitory amplitude was ACA>2-APB.b.ACA and 2-APB preconditioning inhibited sPAs contraction induced by 5-HT in CHPH rats,and the inhibitory extent was ACA>2-APB.c.ACA preconditioning inhibited 5-HT-induced sPAs contraction and intracellular Ca²⁺signal increase（F/F0）in CHPH rats.Conclusion:（1）The dose-dependent contractile response induced by 5-HT in sPAs of CHPH rats is significantly higher than that of normal rats.（2）NOX1/4-TRPM2mediates 5-HT-induced contractile hyperresponsiveness and vascular smooth muscle cell[Ca²⁺]_i changes in sPAs of CHPH rats,in which NOX1 plays a more important role than other subtypes.